Dermal-Epidermal Junction Disruptors: Toxicodynamic Mechanisms

真皮-表皮连接干扰物：毒效机制

基本信息

批准号：
10629516
负责人：
Blase Christopher Billack
金额：
$ 16.4万
依托单位：
ST. JOHN'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-15 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10629516
关键词：
3-Dimensional Achievement Adverse effects Affect Animals Antidotes Antihistamines Antineoplastic Agents Apoptosis Appearance Biological Assay Biopsy Bulla CASP3 gene CASP9 gene Cell Culture Techniques Cells Chemical Warfare Agents Chemicals Chemotherapy-Oncologic Procedure Cicatrix Collagen Collagen Type IV Collagen Type XVII Complement 3d Complex Computer software Cream Cutaneous DNA Data Dermal Dermis Dimethyl Sulfoxide Dose Doxepin Ear Edema Electrolytes Enzyme-Linked Immunosorbent Assay Epidermis Experimental Designs Exposure to Extravasation Formalin Foundations Future Gases Gelatinase B Goals Harvest Histamine Histology Homeostasis Human Immunohistochemistry In Vitro Incidence Infiltration Inflammation Intravenous Kinetics Knockout Mice Learning Leupeptins Lipid Peroxidation Liposomal Doxorubicin Liquid substance Literature Mechlorethamine Mediator Medical Metalloproteases Modeling Mus Mustard Gas Neutrophil Infiltration Nuclear Order Coleoptera Organoselenium Compounds Paclitaxel Pain Paraffin Embedding Patients Peroxidases Pharmaceutical Preparations Poison Predisposition Proteins Reaction Reporting Research Proposals Role Saline Sampling Serine Protease Skin Skin Tissue Snake Venoms Students Supportive care Swelling Techniques Testing Time Tissue Embedding Tissues Topical application Toxic Environmental Substances Toxic effect Tryptase Western Blotting Wild Type Mouse Work World War I analog animal care chemical reduction chemotherapy ebselen experimental study falls human tissue in vivo infection risk inhibitor insight keratinocyte lewisite light microscopy mast cell neutrophil novel protein expression prototype response response to injury skills

3-Dimensional Achievement Adverse effects Affect Animals Antidotes Antihistamines Antineoplastic Agents Apoptosis Appearance Biological Assay Biopsy Bulla CASP3 gene CASP9 gene Cell Culture Techniques Cells Chemical Warfare Agents Chemicals Chemotherapy-Oncologic Procedure Cicatrix Collagen Collagen Type IV Collagen Type XVII Complement 3d Complex Computer software Cream Cutaneous DNA Data Dermal Dermis Dimethyl Sulfoxide Dose Doxepin Ear Edema Electrolytes Enzyme-Linked Immunosorbent Assay Epidermis Experimental Designs Exposure to Extravasation Formalin Foundations Future Gases Gelatinase B Goals Harvest Histamine Histology Homeostasis Human Immunohistochemistry In Vitro Incidence Infiltration Inflammation Intravenous Kinetics Knockout Mice Learning Leupeptins Lipid Peroxidation Liposomal Doxorubicin Liquid substance Literature Mechlorethamine Mediator Medical Metalloproteases Modeling Mus Mustard Gas Neutrophil Infiltration Nuclear Order Coleoptera Organoselenium Compounds Paclitaxel Pain Paraffin Embedding Patients Peroxidases Pharmaceutical Preparations Poison Predisposition Proteins Reaction Reporting Research Proposals Role Saline Sampling Serine Protease Skin Skin Tissue Snake Venoms Students Supportive care Swelling Techniques Testing Time Tissue Embedding Tissues Topical application Toxic Environmental Substances Toxic effect Tryptase Western Blotting Wild Type Mouse Work World War I analog animal care chemical reduction chemotherapy ebselen experimental study falls human tissue in vivo infection risk inhibitor insight keratinocyte lewisite light microscopy mast cell neutrophil novel protein expression prototype response response to injury skills

展开

项目摘要

Project Summary/Abstract Dermal-epidermal junction (DEJ) disruptors are cutaneous poisons that cause the epidermis to detach from the dermis, an effect that results in skin blistering (vesication). The prototype DEJ disruptor is mechlorethamine (MEC), a nitrogen mustard; however, the toxicodynamic mechanisms involved in vesication by MEC and other DEJ disruptors remains mostly undescribed in the scientific literature. The overall goal of the work proposed here is to investigate the toxicodynamic mechanisms associated with DEJ disruption. This will be achieved by studying the role of mast cell derived mediators (histamine and the serine protease tryptase), neutrophil-derived mediators (myeloperoxidase, MPO and matrix metalloproteinase-9, MMP-9) and keratinocyte-specific collagens (collagen IV, COL4 and collagen XVII, COL17) in the response of skin to MEC. In Aim 1 of the proposal, the hypothesis that mast cell-derived mediators influence DEJ disruption will be evaluated. To this end, mouse ear skin will be topically exposed to saline (naïve), dimethylsulfoxide (DMSO, vehicle) or MEC. Additional groups of mice will be treated 20 min prior to exposure to saline, vehicle or MEC with the antihistamine doxepin (5% cream administered topically) or the tryptase inhibitor leupeptin (at test doses of 10 or 30 mg/kg, ip). All ear tissues will be harvested at 2, 8 or 24 hr and either fixed for histopathologic analysis or homogenized and assayed for the presence of histamine via ELISA and tryptase via western blotting. In Aim 2 of the proposal, we will test the hypothesis that neutrophil-derived MMP-9 contributes to DEJ disruption by MEC. To test this hypothesis, we will analyze naïve, vehicle and MEC-treated mouse ear biopsies from wildtype and Mmp9 -/- knockout mice obtained at 2, 8 or 24 hr after exposure for the presence of DEJ disruption using light microscopy and for the presence of myeloperoxidase, a neutrophil-selective marker, using immunohistochemistry (IHC). In Aim 3 of the proposal, we will test the hypothesis that MEC promotes blistering by interfering at the level of epidermal keratinocytes and affecting the expression of key keratinocyte-expressed collagens involved in maintaining the DEJ, namely COL4 and COL17. To test this hypothesis, we will analyze mouse ear biopsies from the time points and samples described above for Aim 1 but here carry out IHC on the formalin-fixed paraffin embedded tissues to evaluate how the expression of these two proteins, COL4 and COL17, changes over time after following exposure to MEC. In addition, experiments in Aim 3 will utilize human 3D skin cultures exposed to MEC for 2, 8 or 24 hr to investigate effects on COL4 and COL17 protein expression in human tissues. All in all, the proposal seeks to investigate three related yet independent hypotheses that will shed new light on the toxicodynamic mechanisms used by DEJ disruptors to cause blisters and will uncover novel targets for the purpose of reducing chemically induced cutaneous blistering. The work proposed here will provide a broad, “whole tissue” toxicodynamic response to injury by MEC at the levels of the cutaneous mast cells, the infiltrating neutrophils and the epidermal keratinocytes and will be complemented by the 3D human skin study. Most importantly, students involved in this project will learn a wide range of skills including experimental design, animal care, animal dosing, histology techniques, IHC, western blotting and how to use ImageJ software to assess tissue protein expression.

项目摘要/摘要皮肤表皮连接（DEJ）破坏者是皮肤的毒药，使表皮从真皮脱落，一个导致皮肤起泡的作用（论文）。原型DEJ破坏者是机电甲胺（MEC），一种氮芥末;但是，MEC和其他DEJ破坏者涉及的毒性机制仍然存在大多在科学文献中没有描述。这里提出的工作的总体目标是调查与DEJ破坏有关的毒性机制。这将通过研究肥大细胞的作用来实现衍生的介体（组胺和序列蛋白胰蛋白酶），中性粒细胞衍生的介质（髓过氧化物酶，MPO和基质金属蛋白酶9，MMP-9）和角质形成细胞特异性胶原蛋白（胶原蛋白IV，COL4和胶原蛋白XVII，COL17）在皮肤对MEC的反应中。在提案的目标1中，肥大细胞衍生介质会影响DEJ的假设将评估中断。为此，小鼠耳朵皮将局部暴露于盐水（幼稚），二甲基硫氧化盐（DMSO，车辆）或MEC。在暴露盐水，媒介物或MEC之前，将对其他小鼠进行其他治疗与抗组胺药Doxepin（局部施用5％奶油）或胰蛋白酶抑制剂亮肽蛋白（以10或10的测试剂量 30 mg/kg，IP）。所有早期组织将在2、8或24小时收获，并固定用于组织病理学分析或通过ELISA和胰蛋白酶通过Western印迹将组胺均质化并分配。在目标2中提案，我们将检验以下假设：中性粒细胞衍生的MMP-9有助于MEC DEJ破坏。测试这个假设，我们将从WildType和MMP9 - / - 敲除分析，媒介物和MEC处理的小鼠耳朵活检暴露于使用光学显微镜和用于DEJ破坏的情况下，在2、8或24小时获得的小鼠使用免疫组织化学（IHC）的存在髓过氧化物酶，一种中性粒细胞选择标记。在目标3中提案，我们将检验以下假设：MEC通过干扰表皮角质形成细胞来促进起泡并影响涉及DEJ的关键角质形成表达胶原蛋白的表达，即Col4和 Col17。为了检验这一假设，我们将从上述时间点和样本中分析小鼠耳朵活检 AIM 1，但在这里在福尔马林固定的石蜡嵌入组织上进行IHC，以评估这两种表达方式暴露于MEC后，蛋白质（Col4和Col17）随着时间的推移而变化。此外，AIM 3中的实验将利用与MEC接触2、8或24小时的人类3D皮肤培养物研究对Col4和Col17蛋白的影响在人体组织中的表达。总而言之，该提案旨在调查三个相关但独立的假设开发了DEJ破坏者引起水泡的毒性动力学机制的新灯，并将发现新的目标为了减少化学诱导的皮肤水泡。这里提出的工作将提供广泛的 MEC在皮肤肥大细胞水平上对损伤的“全组织”毒性动力学反应，浸润中性粒细胞和表皮角质形成细胞将通过3D人类皮肤研究完成。最重要的是，参与该项目的学生将学习广泛的技能，包括实验设计，动物护理，动物剂量，组织学技术，IHC，蛋白质印迹以及如何使用ImageJ软件评估组织蛋白表达。